Biochemical and crystallization analysis of the CENP-SX–DNA complex

CENP-SX is a histone-fold complex that is involved in chromosome segregation and DNA repair. Biochemical and crystallization analysis suggested that multiple molecules of CENP-SX may be involved in DNA binding.


Introduction
Genome integrity is of the utmost importance in all living organisms. Eukaryotes, in particular, undergo mitotic and meiotic cell cycles to proliferate and produce the next generation. Chromosome segregation and DNA repair play pivotal roles in these processes. The CENP-S (MHF1)-CENP-X (MHF2) (CENP-SX) complex (also known as the MHF complex) is a conserved histone-fold complex that participates in these processes (Milletti et al., 2020;Kixmoeller et al., 2020). In chromosome segregation, it forms a complex with another kinetochore component, CENP-T-CENP-W, to form a heterotetrameric CENP-TWSX complex (Nishino et al., 2012). As part of the kinetochore machinery, it connects chromosome and spindle microtubules during mitosis (Kixmoeller et al., 2020). In the absence of the CENP-SX complex, the kinetochore structure becomes abnormal and mis-segregation becomes prominent (Amano et al., 2009). During DNA repair, the CENP-SX complex interacts with FANCM to form the FANCM-CENP-SX complex (Singh et al., 2010;Yan et al., 2010). FANCM is a core component of the Fanconi anemia (FA) pathway and plays a vital role in the localization of the other proteins to the DNA damage site (Milletti et al., 2020).
Biochemical analysis of the purified CENP-SX complex has revealed that it forms a heterotetramer similar to histone H3/H4 (Nishino et al., 2012;Tao et al., 2012). It binds to dsDNA using the histone fold and the basic tail at the C-terminus of CENP-S (Nishino et al., 2012). Interestingly, the binding pattern of the CENP-SX complex to DNA revealed that it forms a regularly spaced protein-DNA complex and the number of proteins increases with the DNA length. Intriguingly, addition of CENP-TW to the CENP-SX-DNA mixture leads to a loss of these regular binding patterns and the CENP-TWSX complex prefers to bind to $100 bp dsDNA. Purified human FANCM-CENP-SX complex has been shown to prefer to bind to branched molecules over dsDNA (Tao et al., 2012;Fox et al., 2014). The low-resolution crystal structure of human CENP-SX in complex with 26 bp dsDNA revealed that CENP-SX uses both its histone-fold and C-terminal basic tail regions in binding to dsDNA . Each CENP-SX dimer was bound to a separate dsDNA duplex and the overall shape resembled a branched DNA molecule. However, the mechanism of regularly spaced DNA binding and branch DNA binding by CENP-SX remains elusive.
Here, using chicken CENP-SX and FANCM-CENP-SX complexes, we tried to perform high-resolution structure analysis of their complexes with DNA. We obtained several crystals of CENP-SX-DNA using different lengths of dsDNA, some of which diffracted to $3.2 Å resolution. These crystals could be separated into two different space groups, each containing multiple molecules in the asymmetric unit. The space group and unit-cell parameters differ from those of the reported complex crystal structures. Thus, determination of the crystal structure should reveal details of the recognition mode. Phase determination and further refinement of the CENP-SX-DNA structure are currently in progress.

Materials and methods
2.1. Macromolecule production and electrophoretic mobility shift assay (EMSA) FANCM-CENP-SX was prepared according to a previous study, replacing truncated CENP-S with full-length CENP-S (Ito & Nishino, 2021; Table 1). CENP-SX was prepared according to a previous report (Nishino et al., 2012). Synthetic oligonucleotides based on the Widom 601 sequence were purchased from Thermo Fisher. Double-stranded DNAs (dsDNAs) were prepared by heat-annealing the complementary oligonucleotides. The dsDNAs were further purified by size-exclusion chromatography in 10 mM Tris pH 7.5, 100 mM NaCl.

Crystallization
To form a protein-DNA complex, a mixture of protein and DNA was incubated at 20 C for 60 min. Initial crystallization screenings for FANCM-CENP-SX-dsDNA were performed using Natrix and Natrix 2 (Hampton Research) by the sittingdrop vapor-diffusion technique in a 96-well format crystallization plate. The final volume of the drop was 0.2 ml, with 0.1 ml of the reservoir solution and the protein-DNA complex, and the plate was incubated at a constant temperature of 20 C.  Table 1 Macromolecule-production information.
The introduction of additional residues, the expression and purification tags, and TEV recognition sites are underlined. Cleavage sites are indicated with a slash.
Conditions for the production of CENP-SX-dsDNA crystals with improved diffraction quality are summarized in Table 2.

Data collection and processing
Diffraction data were collected on BL-1A at the Photon Factory (PF) synchrotron facility (KEK) and were processed with the HKL-2000 package (HKL Research) or XDS (Kabsch, 2010). Data analyses were performed using MOLREP from the CCP4 suite (Winn et al., 2011). Datacollection and processing statistics are summarized in Table 3.

Results and discussion
Chicken and human CENP-SX bind a single dsDNA at regular intervals, whereas human FANCM-CENP-SX prefers branched molecules (Nishino et al., 2012;Fox et al., 2014;. To compare the binding patterns in more detail, we performed EMSA with chicken CENP-SX and FANCM-CENP-SX using synthetic dsDNAs of various lengths   ( Band 2 also started to appear from 25 bp DdsNA and its intensity increased dramatically from 49 bp dsDNA and peaked at 61 bp dsDNA. Interestingly, the intensity of band 2 was stronger and sharper than that of band 1. Band 3 was similar to band 2 and started to appear from 67 bp dsDNA. Band 4 only appeared for 97 bp dsDNA. FANCM-CENP-SX-DNA bands appeared similarly; however, the intensity of band 1 was stronger and sharper than that for CENP-SX-DNA. The other bands were less intense and were smeared. These results suggest that the DNA-binding mode and stoichiometry of CENP-SX differ in the presence and absence of FANCM. To delineate the difference in DNA binding between the two complexes, we performed crystallization experiments in the presence of dsDNA (19, 25, 31, 37, 43 and 49 bp). Irrespective of the length of DNA used, FANCM-CENP-SX-DNA crystals appeared in the presence of 30% 1,4-dioxane. The shapes of the crystals differed according to the length of the DNA (Fig. 2). Rectangular crystals were formed using 19, 25 and 31 bp dsDNA. Needle-shaped crystals appeared using 37, 43 and 49 bp dsDNA. The contents of the crystals were analyzed by two different methods. Addition of DNA-staining  green fluorescent dye to the crystal drop resulted in crystals that glowed green (Fig. 3a). Analysis by SDS-PAGE revealed that the crystals contained CENP-S and CENP-X, whereas FANCM was absent (Fig. 3b). FANCM was present as a film-like structure in the air-liquid interface of the crystal droplet. This situation is similar to a previous report where FANCM was observed to detach from CENP-SX in the presence of organic solvent and oxidative conditions (Ito & Nishino, research communications Acta Cryst.  2021). Thus, FANCM detached from CENP-SX during crystallization even in the presence of DNA. Attempts to reproduce the CENP-SX-DNA crystal using a mixture of CENP-SX and DNA with the same precipitant were unsuccessful and the crystallization conditions were optimized. CENP-SX-DNA crystals appeared in the presence of 40% MPD. The initial crystals diffracted to $7 Å resolution with high mosaicity. Optimization of the DNA and cryoprotectant improved the resolution (Fig. 4). Data analysis showed that there were two different crystals with different space groups and unit-cell parameters. These crystals are both rectangular and are indistinguishable based on their shape. One crystal belonged to space group P2 1 , with unit-cell parameters a = 101, b = 84, c = 112 Å , = 90, = 105, = 90 . The other crystal belonged to space group C2, with unit-cell parameters a = 128, b = 81, c = 100 Å , = 90, = 124, = 90 (Table 3). The volume of the asymmetric units of the two crystals differs by twofold. Matthews analysis of the two crystals indicated that the asymmetric units of the C2 and P2 1 crystals contain $80 000 and $160 000 Da, respectively, with a calculated Matthews coefficient of 2.7 Å 3 Da À1 and a solvent content of 60%. These results suggest that multiple CENP-SX heterodimers and DNA are present in the asymmetric unit. The situation resembles previous low-resolution CENP-SX-DNA complex crystal structures, in which several different crystals were formed and multiple molecules were present in the asymmetric units.
To analyze the relationship between the multiple molecules of CENP-SX and DNA within the crystal, the self-rotation function was calculated. In both the P2 1 and C2 crystals, twofold peaks in the ac plane and fourfold peaks in the 90 plane (Fig. 5) were observed. The twofold symmetry may be due to the symmetry of the CENP-SX tetramer. Alternatively, there may be a twofold-symmetric CENP-SX-DNA complex similar to the reported complex structure, in which CENP-SX dimer-dsDNA complexes were related by twofold symmetry . However, an explanation of the fourfold peak remains elusive. EMSA analyses revealed that multiple CENP-SX tetramers bind to a discrete length of dsDNA. Structure determination should reveal the details of the recognition mechanism. Model building and structure refinement are currently in progress.